By Fluorescence In Situ Hybridization Techniques To Improve Of Cholangiocarcinoma Clinical Diagnosis And Mechanism

BackgroundThe bile duct carcinoma is a neoplasm of the bile duct epithelium, which accounts for about 15% of hepatobiliary cancers. The preoperative diagnosis of the bile duct carcinoma mainly depends on histocytology, because masses in the biliary tract can seldom be revealed by sectional imaging. However, the diagnostic value of bile duct cytology is comparatively low, as the bile duct is not readily accessible in anatomy and the quantity of biopsied material from the bile duct is limited. Therefore, more sensitive and reliable diagnostic methods for the bile duct carcinoma are required. Anueploidy is a phenomenon which is widely presented in tumor cells of the human being. Fluorescent in situ hybridization (FISH) has been used to detect anueploidy in tumors, but there were limited reports to evaluate its diagnostic value in the bile duct carcinoma. Aurora kinase A (AURKA) is an important regulator in mitosis of the cell. Its overexpression is often associated with unequal segregation of the chromosome and anueploidy. It has been proved that AURKA is overexpressed in many tumors of the human being and associated with the phenotype of tumors, while the expression of AURKA in the bile duct carcinoma is not known.ObjectivesBy studies of cell lines, tissues and brushing samples of the bile duct carcinoma, we're going to understand 1) The frequency of anueploidy in the bile duct carcinoma; 2) The diagnostic value of FISH (UroVysion kit) for the bile duct carcinoma in brush samples; 3) The expression of AURKA in the bile duct carcinoma; 4) The association between AURKA expression and phenotype of the bile duct carcinoma.MethodsUroVysion kit was chosen for FISH in this study, as it has been widely used in cytological studies and approved by FDA. The anueploidy was detected by FISH in biliary cell lines of QBC939, GBC-SD, and compared with that in the cell line of CC-HEL-1 and cells from the gallbladder epithelium and hepatic cells, in tissues of the bile duct carcinoma and compared with that in inflammatory tissues. Furthermore, the diagnostic value of FISH was assessed in 27 brush samples of biliary cytology and compared with that of routine cytology (RC), with the gold standard established by the pathological diagnosis or clinical outcome after 6-months follow-up. The expression of AURKA in biliary cell lines of QBC939, GBC-SD, CC-HEL-1, fresh frozen bile duct epithelium and hepatic cells was measured by Real-Time PCR and Western Blotting. 29 samples of the bile duct carcinoma and 8 samples of the biliary inflammatory tissue were also studied by immunohistological stain for the expression of AURKA. The association of the expression level of AURKA and the tumor stage and grade was analyzed.ResultsAnueploidy is frequently presented in cells of biliary cancers both in vivo and in vitrosamples by FISH, while it is rarely seen in benign cells and tissues of the bile duct. Thecytological diagnostic value of the bile duct carcinoma was improved by FISH(UroVysion) detecting locus in chromosome 3, 7, 17 and 9p21 in brush samples. Thesensitivity, specificity, positive predictive value and negative predictive value are 50%,100%, 100%, and 31.3% respectively by FISH, and are 22.7%, 100%, 100%, and 22.7%respectively by RC, p=0.146. Polysomy of chromosome 3 is the major type ofanueploidy in the study, and homogeneous loss of 9p21 can also be seen.The expression of AURKA in biliary malignant cell lines is significantly higher than inbenign one. Immunohistological stain showed that AURKA was overexpressed in biliarycarcinomas, and the relative level of expression of AURKA is also associated with thetumor stages and grades.Conclusion: Anueploidy is frequently presented in cells of the bile duct carcinoma. FISHcan improve the cytological diagnostic value for biliary cancers. AURKA isoverexpressed in the bile duct carcinoma, and the expression level is associated with thetumor phenotype.